Fig. 4

1 The characterization of neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). In panel (a), the process of generating NS/PCs from feeder-free cultured hiPSCs is depicted, accompanied by representative images of cells at each stage of differentiation. The scale bar in the image is 200 μm. Panels (b) and (c) show representative immunocytochemical images (b) and quantification (c) of hiPSC-NS/PCs using specific antibodies against SOX1, SOX2, and NESTIN. The inset in panel (b) displays Hoechst nuclear staining of the same sample, and the scale bar is 50 μm. Panel (d) presents representative images of cell surface markers PSA-NCAM and CD133 on hiPSC-NS/PCs. The differentiation capacity of hiPSC-NS/PCs is demonstrated in panel (e) with representative images of neuronal differentiation for each cell line. Neuronal markers, including MAP2ab (green), NeuN (red), and βIII-tubulin (purple), are expressed after 14 days of differentiation. The scale bar in the image is 100 μm. Panel (f) shows histological evaluation of hiPSC-NS/PCs after transplantation into immunodeficient mice. Representative tissue sections of the striatum are displayed, and graft survival is assessed using the marker STEM121, which indicates human cytoplasm. The differentiation capacity of hiPSC-NS/PCs in the graft is evaluated using antibodies against Ki67, NESTIN, and human-specific GFAP (STEM123). Insets in the panel provide a closer look at the Ki67 signal in specific regions. The scale bars in the images are 500 μm. Panel (g) quantifies the number of Ki67 + cells among human-specific Lamin A + C + cells at the indicated time point. Panels (h) and (i) demonstrate neuronal differentiation of hiPSC-NS/PCs after transplantation, as indicated by the expression of the neuronal marker nELAVL in HNA + grafts. The insets in panel (h) show Hoechst nuclear staining of the same sample, and the scale bar is 20 μm. Quantification of neuronal differentiation is shown in panel (i). Statistical values are provided as means ± standard deviation, and asterisks indicate statistical significance (NS/PC-A, n = 3; NS/PC-B, n = 3; EB-NS/PC, n = 4, **p < 0.01). 2 The examination of the variability within hiPSC-NS/PCs (human induced pluripotent stem cell-derived neural stem/progenitor cells) using a single-cell-based method. a) The diagram illustrates the process of fluorescence-activated cell sorting (FACS) of NS/PC-B, followed by cell expansion for subsequent biological analyses. b) The figure displays a correlation analysis between gene expression profiles of single-cell-derived NS/PCs (scNS/PCs) obtained through microarray analysis and gene expression in NS/PCs, neural crest cells (NCCs), and MSCs from publicly available datasets. The clustering of these profiles is also presented, with the color indicating the significance of correlation (z-value). c) Principal component analysis of scNS/PCs is shown. NS/PC-like scNS/PCs are represented by red dots, NCC-like scNS/PCs by blue dots, and unclassified scNS/PCs (intermediate scNS/PCs) by light green dots. d) A comparison of gene expression related to neural (NES, SOX2, and ZBTB16) and mesodermal (SOX9 and PDGFR) lineages is demonstrated in NS/PC-like (blue), intermediate (light green), and NCC-like (red) scNS/PCs. e) The figure presents a gene ontology (GO) analysis of differentially expressed genes in NS/PC-like scNS/PCs compared to NCC-like scNS/PCs. 3 The presence of cells displaying mesodermal characteristics in grafts derived from human-induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs). In panel (a), the grafts in the striatum were examined histologically using antibodies against SOX1 and SOX9, and the arrows highlight cells that are positive for SOX1 and SOX9 among the HNA-positive cells. The scale bar represents a length of 25 μm. Panel (b) provides a quantification of the SOX1-SOX9 positive cells in the grafts, with the mean values and standard deviations indicated [NS/PC-A (3M) n = 3; NS/PC-B (3M) n = 3; NS/PC-B (6M) n = 4, *p < 0.05]. In panel (c), representative images show the expression of AP2α in the NS/PC-derived grafts in the striatum, with the inset demonstrating Hoechst nuclear staining of the same field. Panel (d) quantifies the frequency of AP2α-positive cells in the grafts, with mean values and standard deviations provided [NS/PC-A (3M) n = 4; NS/PC-B (3M, 6M) n = 4, **p < 0.01]. Panel (e) displays representative images of Vimentin and SNAI1 expression in STEM121-positive grafts six months after transplantation into an injured spinal cord. The scale bar represents a length of 100 μm. Panel (f) presents a bone-like structure derived from the grafts in the injured spinal cord region. Immunohistochemical staining of Ki67 (upper panel) and H&E staining (lower panel) of serial sections corresponding to the area shown in (e) is shown. The inset provides a higher magnification of the boxed field. The scale bar represents a length of 100 μm. 4 The osteogenic differentiation capacity of neural stem/progenitor cells (NS/PCs) that have characteristics resembling neural crest cells (NCCs). Panel (a) provides detailed information about the cluster numbers within NS/PCs and NCC-like NS/PCs, with the selected cells for further analysis highlighted in red. Panel (b) shows a principal component analysis (PCA) plot of the transcriptome in the NS/PCs, with additional information about the selected NS/PCs highlighted in blue (NS/PC-like) and red (NCC-like). Panel (c) presents representative images of the selected NS/PCs, with a scale bar of 100 μm for size reference. Panel (d) displays the results of immunocytochemical analysis of the NS/PC-like and NCC-like NS/PCs using antibodies against SOX1 (green), SOX9 (red), and NESTIN (purple). The inset in this panel shows Hoechst nuclear staining of the same field, with a scale bar of 50 μm. Panel (e) provides quantification data based on the immunocytochemical analysis shown in panel (d). Finally, panel (f) shows the results of Alizarin red S staining after osteogenic differentiation of the NS/PC-like and NCC-like NS/PCs. The scale bar in this panel is 100 μm. 5 The process of identifying specific cell surface markers to determine populations that possess the ability to generate bone tissue. In part (a), a screening was conducted on a subset of hiPSC-NS/PCs (human-induced pluripotent stem cell-derived neural stem/progenitor cells) using the BD Lyoplate screening panel. The results from flow cytometry categorized the antibodies into three groups. Part (b) shows the flow cytometric analysis of cell surface markers for pluripotent stem cells (PSCs), NS/PCs, and MSCs on NS/PC-B cells. Part (c) validates the cell surface marker screening using NS/PC-like and NCC-like scNS/PCs (single-cell-derived NS/PCs and neural crest cell-like NS/PCs). Flow cytometric analysis displays the frequencies of cells expressing the antigens. Part (d) provides a representative image of coexpression analysis between NS/PC markers and NCC markers on NS/PC-like and NCC-like scNS/PCs. In part (e), the expression of NCC markers on various types of iPSC-NS/PCs is evaluated, along with representative images of Alizarin red S staining after inducing osteogenic differentiation. Part (f) involves sorting NS/PC-B cells based on CD15, CD73, and CD105 expression. The sorted cells are then subjected to further evaluation. Part (g) quantifies the sorted fractions based on SOX1 and SOX9 expression. Part (h) examines the sorted cells for their ability to differentiate into bone cells using Alizarin red S staining. Finally, part (i) presents a proposed model for the cellular heterogeneity of hiPSC-NS/PCs. 6 The transcriptome characteristics of iPSC-NS/PCs in comparison to authentic NCCs and MSCs. In panel (a), a heatmap demonstrates the expression levels of genes associated with NS/PCs (SOX1 and NES) and genes associated with NCCs (SOX9, SOX10, AP2α, and FOXD3) in parental NS/PCs, scNS/PCs, hiPSC-NCCs, and MSCs. Panel (b) displays a heatmap that shows the correlation in gene expression between parental NS/PCs and scNS/PCs with gene expression data from previously published datasets of PSA-NCAM + and PSA-NCAM- NS/PCs. The color scale represents the z-value, indicating the significance of the correlation. In panel (c), a principal component analysis is presented, comparing scNS/PCs with referenced cells such as hiPSC-NCCs, WBM, and MSCs. 7 The process of ensuring the quality of neural stem/progenitor cells (NS/PCs) through purification using CD15. In panel (a), a diagram shows the transplantation of NS/PCs derived from NS/PC-B, either sorted with an anti-CD15 antibody [sorting ( +)] or without it [sorting (-)], into the striatum of immunodeficient mice. After 10 weeks, immunohistochemical analysis was conducted to evaluate the differentiation capacity of the transplanted NS/PCs. Representative images (b) and corresponding quantification (c) demonstrate the expression of AP2α in HNA + grafts as an indicator of differentiation capacity. The insets in panel (b) display Hoechst nuclear staining of the same area. Quantitative data is presented in the right panel. The scale bar represents 50 μm. Mean values ± standard deviation (n = 3, *p < 0.05) are provided. Similarly, representative images (d) and quantification (e) show the expression of nELAVL in HNA + grafts to assess the differentiation capacity of the transplanted NS/PCs. The insets in panel (d) display Hoechst nuclear staining of the same area. Reprinted from [84] with permission from the Springer Nature